1.1 Field of the Invention
This invention relates to volatile photoabsorbing matrices having a low sublimation temperature for use in the mass spectrometric analysis of large, nonvolatile molecules. This invention also relates to methods for preparing samples containing large, nonvolatile analyte molecules for laser desorption mass spectrometry employing such matrices.
1.2 Description of Related Art
Approximately 4,000 human disorders are attributed to genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the Human Genome Project is to find all genes associated with each disorder. The definitive diagnostic test for any specific genetic disease (or predisposition to disease) will be the identification of polymorphic variations in the DNA sequence of affected cells that result in alterations of gene function. Furthermore, response to specific medications may depend on the presence of polymorphisms. Developing DNA (or RNA) screening as a practical tool for medical diagnostics requires a method that is inexpensive, accurate, expeditious, and robust.
Genetic polymorphisms and mutations can manifest themselves in several forms, such as point polymorphisms or point mutations where a single base is changed to one of the three other bases; deletions where one or more bases are removed from a nucleic acid sequence and the bases flanking the deleted sequence are directly linked to each other; insertions where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the overall sequence; and expansions and reductions of repeating sequence motifs. Large insertions and deletions, often the result of chromosomal recombination and rearrangement events, can lead to partial or complete loss of the activity of a gene. Of these forms of polymorphism, in general the most difficult type of change to screen for and detect is the point polymorphism because it represents the smallest degree of molecular change.
Although a number of genetic defects can be linked to a specific single point mutation within a gene, e.g. sickle cell anemia, many are caused by a wide spectrum of different mutations throughout the gene. A typical gene that might be screened could be anywhere from 1,000 to 100,000 bases in length, though smaller and larger genes do exist. Of that amount of DNA, only a fraction of the base pairs actually encode the protein. These discontinuous protein coding regions are called exons and the remainder of the gene is referred to as introns. Of these two types of regions, exons often contain the most important sequences to be screened. Several complex procedures have been developed for scanning genes in order to detect polymorphisms. These procedures are applicable to both exons and introns.
In terms of current use, most of the methods to scan or screen genes employ slab or capillary gel electrophoresis for the separation and detection step in the assays. Gel electrophoresis of nucleic acids primarily provides relative size information based on mobility through the gel matrix. If calibration standards are employed, gel electrophoresis can be used to measure absolute and relative molecular weights of large biomolecules with some moderate degree of accuracy; even then, the accuracy is typically only 5% to 10%. Also the molecular weight resolution is limited. In cases where two DNA fragments with the identical number of base pairs can be separated, for example, by using high concentration polyacrylamide gels, it is still not possible to identify which band on a gel corresponds to which DNA fragment without performing secondary labeling experiments. Thus, gel electrophoresis techniques can only determine size and cannot provide any information about changes in base composition or sequence without performing more complex sequencing reactions. Gel-based techniques, for the most part, are dependent on labeling or staining methods to visualize and discriminate between different nucleic acid fragments.
Many methods in use today capable of screening broadly for genetic polymorphisms suffer from technical complication and are labor and time intensive. Single strand conformational polymorphism (SSCP) (Orita et al., 1989), denaturing gradient gel electrophoresis (DGGE) (Abrams et al., 1990), chemical cleavage at mismatch (CCM) (Saleeba and Cotton, 1993), enzymatic mismatch cleavage (EMC) (Youil et al., 1995), and cleavage fragment length polymorphism (CFLP) procedures are currently gel-based, making them cumbersome to automate and perform efficiently. Thus, there is a need for new methods that can provide cost effective and expeditious means for screening genetic material in an effort to detect genetic mutations and diagnose related medical conditions simply, quickly, accurately, and inexpensively.
Another approach that is having some success is to employ mass spectrometry to screen for and detect genetic mutations as well as to sequence nucleic acids. In order to measure the mass of nonvolatile high molecular weight molecules, typically greater than 1000 Da, in a mass spectrometer, the analyte molecules must first be volatilized or converted into gas-phase ions. Although direct laser desorption of the neat analyte is one approach to volatilizing the molecule, the energy deposited into the analyte may induce fragmentation and lead to results that are ambiguous or difficult to analyze. The late 1980's saw the rise of two new mass spectrometric techniques which are potentially suitable for genetic screening tests by successfully measuring the masses of intact very large biomolecules, namely, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) (Tanaka et al., 1988; Spengler et al., 1989) and electrospray ionization (ES) combined with a variety of mass analyzers. The MALDI mass spectrometric technique can also be used with methods other than time-of-flight, for example, magnetic sector, Fourier-transform ion cyclotron resonance, quadrupole, and quadrupole trap.
MALDI-TOF MS involves laser pulses focused on a small sample plate on which analyte molecules (i.e. nucleic acids) are embedded in either a solid or liquid matrix which is typically a small, highly absorbing material, such as a small aromatic organic molecule. The volatilization of intact fragile molecules benefits from the use of matrix-assisted laser desorption ionization because the radiative energy from the laser pulse is coupled indirectly into the analyte through the matrix molecules. Typically, the analyte molecules are crystallized with a large molar excess of a photoabsorbing matrix (see U.S. Pat. Nos. 4,920,264 and 5,118,937, incorporated herein by reference). An advance in MALDI analysis of polynucleotides was the discovery of 3-hydroxypicolinic acid (3-HPA) as a suitable matrix for mixed-base oligonucleotides (Wu, et al., 1993).
The laser pulses transfer energy to the matrix causing a microscopic ablation and concomitant ionization of the analyte molecules, producing a gaseous plume of intact, charged nucleic acids in single-stranded form. It is thought that upon laser excitation the matrix molecules are rapidly heated and ejected into the gas phase, carrying analyte molecules into the expansion plume of molecules and ions. It is thought that gas-phase ion-molecule collisions subsequently ionize the neutral analyte molecules in the near-surface region, often via proton transfer. The matrix thus functions as both an energy- and charge-transfer species. If double-stranded nucleic acids are analyzed, the MALDI-TOF MS typically results in detection of mostly charged denatured single-stranded nucleic acids.
The ions generated by the laser pulses are accelerated to a fixed kinetic energy by a strong electric field and then passed through an electric field-free region in vacuum, traveling with a velocity corresponding to their respective mass-to-charge ratios (m/z). Thus, the smaller m/z ions will travel through the vacuum region faster than the larger m/z ions thereby causing a separation. At the end of the electric field-free region, the ions collide with a detector that generates a signal as each set of ions of a particular mass-to-charge ratio strikes the detector. Usually for a given assay, 10 to 100 mass spectra resulting from individual laser pulses are summed together to make a single composite mass spectrum with an improved signal-to-noise ratio.
The mass of an ion (such as a charged nucleic acid) is measured by using its velocity to determine the mass-to-charge ratio by time-of-flight analysis. In other words, the mass of the molecule directly correlates with the time it takes to travel from the sample plate to the detector. The entire process takes only microseconds. In an automated apparatus, tens to hundreds of samples can be analyzed per minute. In addition to speed, MALDI-TOF MS has one of the largest mass ranges for mass spectrometric devices. The current mass range for MALDI-TOF MS is from 1 to 1,000,000 Da (measured recently for a protein) (Nelson et al., 1995).
The performance of a mass spectrometer is measured by its sensitivity, mass resolution and mass accuracy. Sensitivity is measured by the amount of material needed; it is generally desirable and possible with mass spectrometry to work with sample amounts in the femtomole and low picomole range. Mass resolution, m/.DELTA.m, is the measure of an instrument's ability to produce separate signals from ions of similar mass. Mass resolution is defined as the mass, m, of an ion signal divided by the full width of the signal, .DELTA.m, usually measured between points of half-maximum intensity. Mass accuracy is the measure of error in designating a mass to an ion signal. The mass accuracy is defined as the ratio of the mass assignment error divided by the mass of the ion and can be represented as a percentage.
To be able to detect any point polymorphism directly by MALDI-TOF mass spectrometry, one would need to resolve and accurately measure the masses of nucleic acids in which a single base change has occurred (in comparison to the wild type nucleic acid). A single base change can be a mass difference of as little as 9 Da. This value represents the difference between the two bases with the closest mass values, A and T (A=2'-deoxyadenosine-5'-phosphate=313.19 Da; T=2'-deoxythymidine-5'-phosphate=304.20 Da; G=2'-deoxyguanosine-5'-phosphate=329.21 Da; and C=2'-deoxycytidine-5'-phosphate=289.19 Da). If during the mutation process, a single A changes to T or a single T to A, the mutant nucleic acid containing the base transversion will either decrease or increase by 9 Da in total mass as compared to the wild type nucleic acid. For mass spectrometry to directly detect these transversions, it must therefore be able to detect a minimum mass change, .DELTA.m, of approximately 9 Da.
For example, in order to fully resolve (which may not be necessary) a point-mutated (A to T or T to A) heterozygote 50-base single-stranded DNA fragment having a mass, m, of .about.15,000 Da from its corresponding wild type nucleic acid, the required mass resolution is m/.DELTA.m=15,000/9.apprxeq.1,700. However, the mass accuracy needs to be significantly better than 9 Da to increase quality assurance and to prevent ambiguities where the measured mass value is near the half-way point between the two theoretical masses. For an analyte of 15,000 Da, in practice the mass accuracy needs to be .DELTA.m.about..+-.3 Da=6 Da. In this case, the absolute mass accuracy required is (6/15,000)*100=0.04%. Often a distinguishing level of mass accuracy relative to another known peak in the spectrum is sufficient to resolve ambiguities. For example, if there is a known mass peak 1000 Da from the mass peak in question, the relative position of the unknown to the known peak may be known with greater accuracy than that provided by an absolute, previous calibration of the mass spectrometer.
In addition, the ability to separate DNA fragments (1) differing in only one base in length and (2) of reasonable length (e.g, of sizes corresponding to at least primer size, around 20 to 30 bases or so up to about 50 bases in length) is critical to achieving even rudimentary DNA sequencing by MALDI-MS. For laser desorption mass spectroscopy techniques to successfully analyze macromolecules requires that one stably laser-desorb molecules into a vapor phase, and separate and detect (and thereby determine the mass of) the volatilized molecules by mass spectroscopy. The ability to stably desorb the macromolecule depends on the availability of a suitable light absorbing matrix that will allow one to stably laser-desorb DNA molecules from a solid state to a gaseous state, and permit separation of DNA molecules having only a nucleotide or so difference in length. Putting that into perspective, the difference in mass between a polynucleotide having 30 versus 31 nucleotide represents about a 3% difference in mass (about 9610 v. 310, assuming an average m.w. of 310 for each nucleotide). If one applies this to a DNA molecule of 100 nucleotides in length, a modest sequence by DNA sequencing standards, the separation system must distinguish among DNA molecules differing by only 1% in mass.
Thus, there is a need for the development of MS techniques and related materials for practicing these techniques that have enhanced resolution, accuracy, and sensitivity. The ability to stably desorb the molecule from a solid matrix that absorbs light at the laser wavelength, without radiation damage and fragmentation of the sample is particularly important as fragmentation can lead to complex spectra and decreased resolution and sensitivity.
Although MALDI generates less energetic analyte ions than direct laser desorption, thus decreasing the thermal degradation of the analyte, the ions nevertheless contain significant internal energy, which may result in fragmentation. Among the few matrix molecules that have been found to desorb/ionize intact DNA, 3-HPA is currently the most widely used (Wu et al., 1993; Wu et al., 1994)). Using a matrix mixture of 3-HPA with picolinic acid, oligonucleotides have been detected that are greater than 500 bases (up to about 200 kDa) in length (Tang et al., 1994; Liu et al., 1995). However, as the length of the oligonucleotide increases, the mass resolution is degraded by widening kinetic energy spreads, prompt fragmentation, delayed fragmentation (metastable decay), and the formation of matrix adducts. Thus, there is a need to develop MS materials and methods that minimize fragmentation of the analyte ions during the MALDI process, extend the accessible mass range for mass spectrometric detection, and enhance the utility of the MS techniques.